Thrombocytopenia affects 20-30% of infants admitted to the neonatal intensive care unit. In 25% of affected neonates (25,000-30,000 per year in the US), the thrombocytopenia is severe, and 9% of them experience clinically significant bleeding (usually intracranial). Up to 10% of severely thrombocytopenic neonates have low platelet counts lasting >30 days, prompting multiple platelet transfusions, with all their associated risks. The causes underlying the predisposition of neonates to develop thrombocytopenia are not understood, but are clearly related to the unique biological characteristics of fetal/neonatal megakaryocytes (MKs). However, there is a fundamental gap in understanding the mechanisms underlying the differences between neonatal and adult MKs, and lack of such knowledge has impaired our ability to treat neonatal thrombocytopenia and other disorders that selectively affect fetal/neonatal MK progenitors. Over the last period of support, we discovered that fetal megakaryopoiesis is characterized by a unique uncoupling of proliferation, polyploidization, and cytoplasmic maturation, and that cell-intrinsic and micro-environmental factors contribute to this pattern. Specifically, our data suggest that a pathway involving the miRNA let-7b mediates their high proliferative rate. Furthermore, we found that neonatal MK progenitors respond differently to thrombopoietin (Tpo) compared to adults and exhibit differences in Tpo receptor expression and signaling pathways. This finding is of high clinical significance, given the recent FDA approval of 2 Tpo mimetics (Romiplostim and Eltrombopag) for the treatment of thrombocytopenic adults. While some thrombocytopenic neonates are appealing candidates for treatment with these mimetics, our data suggest that simply extrapolating observations from adults to neonates would represent a serious pitfall. The objectives of the current proposal are two-fold: 1) to advance our understanding of the mechanisms underlying the unique features of fetal/neonatal megakaryopoiesis; and 2) to characterize the responses of neonatal MK progenitors to these novel Tpo agonists, with the long term goal of improving the treatment of thrombocytopenic neonates. To achieve our objectives, we designed the following Specific Aims: 1) To dissect the contributions of cell intrinsic and microenvironmental factors (Tpo and others) on the phenotype of fetal/neonatal megakaryopoiesis; 2) To characterize the effects of romiplostim and eltrombopag on fetal, neonatal, and adult human megakaryocytopoiesis; and 3) To establish the platelet production rate at baseline and in response to romiplostim in newborn mice with thrombocytopenia. These aims will be accomplished through a blend of in vitro studies on primary human MKs and in vivo studies using knockout mice and murine models of thrombocytopenia. We expect our findings to have a substantial impact on both our fundamental understanding of fetal MK biology and on the clinical management of thrombocytopenic neonates.